PROJECT SUMMARY Repetitive satellite DNA is a major component of most eukaryotic genomes, including humans. Satellite DNAs can exist as selfish genomic parasites that propagate in the genome at the expense of the host, or, alternatively, may form essential chromosome structures including centromeres, which ensure proper chromosome separation during cell division, and telomeres, which protect the ends of chromosomes from degradation. Despite participating in these essential structures, satellite DNA sequences differ widely in sequence and abundance in the genome, even between closely related species. Molecular and theoretical models exist to explain how and why repetitive DNA shows such rapid evolution. However, few studies test these models using genome-wide data because the repetitive part of the genome is difficult to accurately sequence and measure with current technology. It is important to understand how repeats vary across populations, how and why they change over time, and their underlying genetic determinants because they are associated with diseases affecting human health including cancer, differences in immunity, and premature aging. To understand the evolutionary processes behind and the genetic basis of repeat variation, experimental systems are needed in which variation in satellite DNA has been documented across populations and species and can be assayed for phenotypic effects. Drosophila melanogaster has been a workhorse of genetic research for over a century and genomic short-read datasets are readily available for over 1000 lines of D. melanogaster and ~50 lines of three closely related species. I propose to harness the power of these genomic resources to study the evolutionary dynamics and genetic basis of satellite DNA. First, I will assay this dataset for satellites using a customized software pipeline that accounts for GC-bias, currently a major confounding factor in accurate assessment of genome-wide satellite abundance in short-read data. Second, I will use this data to inform tests of two hypotheses for rapid satellite turnover: neutral evolution and selfish transmission. If satellites are evolving neutrally, then variation across populations and species should correlate with divergence in a comparative analysis. If satellites are evolving rapidly due to selfish transmission, then I will detect significant deviations from Mendelian genotype ratios in backcrosses between D. melanogaster lines that differ in satellite content. Finally, I will investigate the genetic basis for satellite abundance using genome- wide associations (GWA), both for individual sequences and satellites in aggregate. If satellite abundance has a simple genetic basis, then GWA analysis will show significant associations with only a few genetic variants. This work will set the standard for detection and assessment of satellite DNA in short-read datasets, provide insight into modes of genome-wide satellite evolution, estimate the strength and frequency of selfish transmission, and generate candidate genes associated with satellite abundance, thus enabling a major advance in our understanding of the evolutionary dynamics and genetic determinants of satellite DNA.